Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein.
Flp is a member of the integrase family of site-specific recombinases. Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget). We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity. These single-stranded oligonucleotides differ in sequence from the duplex FRT site. The minimal length of the oligonucleotides which is active is 29 nt. This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides. Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element. We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp.
Zhu
X D
XD
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Sadowski
P D
PD
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase